Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer

Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer Yajing Zhao, Yapeng Zhao, Lisheng Liu, Guanghao Li, Yawen Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4013931/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Tumor-exosomal miRNA play crucial roles in cancer diagnosis. The current reports aimed to found an exosomal miRNA for colorectal cancer(CRC) with non-invasiveness, sensitivity and specificity. Patients and methods: The exosome was extracted from CRC patients and healthy donors using ultracentrifugation to verify by TEM, qNano and western blot. Differential expression level and clinical characterization of miR-205-5p were analyzed in colorectal cancer via TCGA. Real Time Quantitative PCR was employed to determine the different expression levels of exosomal miRNAs in 157 primary CRC patients and 135 healthy donors. Predictions were made concerning target genes to determine the direction for further exploring the etiopathogenesis of the disease by means of bioinformatics. Results: The expression of miR-205-5p demonstrated a substantial downregulation in colorectal cancer compared to healthy donors, as determined through analysis of the TCGA database. We conducted a prediction and analysis of the functional enrichment of downstream target genes regulated by miR-205-5p. Reduced level of exosomal miR-205-5p in serum from CRC patients was found compared with healthy controls (p<0.0001, respectively) and benign disease (p<0.0001, respectively). The levels of expression of exosomal miR-205-5p were substantially lower in early stage CRC patients than in the contrast groups (p<0.0001, respectively). The expression levels of exosomal miR-205-5p saw significant increase postoperatively (p=0.0053, respectively). Conclusions: The present study demonstrated that serum exosomal miR-205-5p may be a diagnostic biomarker for colorectal cancer. exosomes colorectal cancer miR-205-5p diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) ranks third among the commonest cancers and poses a big threat to people’s health and life. Nevertheless, approximately four out of ten CRC patients end up with relapse and recurrent metastasis, and merely a small portion of them have a survival time of five years ( 1 , 2 ). Hence, the development of effective and easy-to-use screening tools is of vital importance for early diagnosis and precise stage classification in CRC patients. These may include fluid biopsy such as proteins, tumor markers, circulating tumor cells( 3 ), cell-free DNA( 4 ), and exosomes( 5 ), to facilitate the early detection and precise stage classification of CRC( 6 ). Exosomes, also referred to as extracellular vesicles, are secreted organelles with a single membrane and measure 30 to 200 nm in diameter ( 7 ). Cells communicate via exosomes ( 8 ). Therefore, as an essential messenger between cells, exosomes transmit signals to neighbouring cells or to distant anatomical sites by carrying cytokines ( 9 ), modify proteic and genetic expression in receiving cells, and thus influence the functions of receiving cells. Exosomes play multi-roles as well in the pathogenesis ( 10 ) metastasis ( 11 ) and immunity ( 12 ). Exosomes encompass a variety of proteins ( 13 ), miRNAs ( 14 ) and DNA( 15 ), which are apt to be transferred to target cells. For instance, extensive deregulation of miRNAs have shown in human cancers, which highlights their key role in the onset, development and metastasis of the tumor, and hence miRNAs become potential biomarkers for cancers. MicroRNA (miRNA) are tiny, non-coding RNA which are generally 20–25 nucleotide long sequences( 16 ), and which will unite with the target mRNA sequence after they enter the receptor cell ( 17 ). A host of studies have demonstrated that exosomal miRNA is capable of mediating communication between cells, thus involved in tumor carcinogenesis, metastasis, immunosuppression, angiogenesis, and other aspects ( 18 , 19 ). Emerging evidence suggested that five types of exosomal miRNAs, namely miR-205, miR-19a, miR-19b, miR-30b and miR-20a, are promising diagnostic markers of squamous cell lung cancer due to their decreased levels of circulation after resection operation of the lesion ( 20 ). The level of serum exosomal miR-205 was up-regulated in ovarian cancer tissues, and its over-expression was linked to cancerous metastasis in ovarian carcinoma patients ( 11 ). The relative expressions of exosomal miR-1246 in serum were signifcantly overexpressed in gastric cancer patients compared to that in healthy controls ( 21 ). Nasopharyngeal carcinoma cells discharge exosomes that contain certain types of miRNAs that play an inhibitory role in T-cell proliferation,targeting MAPK-1 and STAT pathways ( 22 ). The increase of neutrophil count in peripheral blood has been shown to be related to poor clinical outcomes in pancreatic cancer, gastric cancer and breast cancer. Rao et al demonstrated that the increase in intertumoral neutrophils was associated with malignant phenotypes and could predict adverse prognosis in CRC. This study aimed to verify the different expression levels of serum exosomal miRNA in CRC patients and in contrast groups by means of small RNA sequencing and fluorescence quantitative PCR. Consequently, exosomal miR-205-5p were chosen and their related with diagnostic efficiency and clinical features was analyzed. Therefore, exosomal miR-205-5p as a novel diagnostic biomarker. Materials and Methods 2.1 Patients and clinical samples In the study, we selected altogether 157 CRC patients, 135 healthy donors and 20 benign disease patients hospitalized at the Shangdong Caner Hospital in September 2017 to July 2018. We obtained their written consent. The estimation of tumor staging was performed in accordance with the AJCC Cancer Staging Handbook, 2017. The protocol was approved by the Shandong Cancer Hospital Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences of committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki. No patient was given anti-tumor treatment prior to serum collection and no patient was diagnosed with diseases of the endocrine, immune or metabolic system. Sera collection was performed on 17/157 patients with CRC, who received surgical treatment after two months. Patients’ clinical features and diabetic history were investigated (Table 1 ). Table 1 Characteristics of CRC patients for differentially expressed exosomal miR-205-5p miR-205-5p Characteristic No. cases Median with interquartile range P-value Age(year) <61 78 4.4729(4.0255–4.9203) 0.954 ≥ 61 79 4.4001(3.9076–4.8925) Gender Male 105 4.6445(4.2411–5.0479) 0.094 Female 52 4.0158(3.4443–4.5872) Drinking status Yes 28 4.0204(3.0903–4.9504) 0.181 No 129 4.5265(4.1756–4.8774 Diabetes status Yes 19 4.3734(3.2583–5.4885) 0.951 No 138 4.4449(4.0973–4.7924) Tumor position rectum 92 4.4328(3.9982–4.8674) 0.920 colon 65 4.4411(3.9231–4.9591) Tumor size <30 50 4.5427(3.9112–5.1742) 0.748 ≥ 30 60 4.3591(3.8134–4.9048) unknown 47 Lymph node metastasis status Yes 62 4.3886(3.9132–4.8640) 0.405 No 72 4.6332 (4.1118–5.1546) unknown 23 Distant metastasis Yes 35 4.1779(3.4966–4.8591) 0.386 No 122 4.5104(4.1304–4.8903) 2.2 Isolation of exosomes Serum underwent ultracentrifugation ( 23 ) under the condition of 10000 × g for 30 min at 4°C for the purpose of removing the cellular debris, and then under the condition of 100,000 × g for 2 h at 4°C for exosome precipitation. Then, the samples were analyzed using the following techniques. 2.3 TEM assay Transmission electron microscopy (TEM) was conducted for the purpose of identifying the purified exosomes. The exosome pellets were moved to the grids of solution (a 50 µL drop of 1% glutaraldehyde), and after a standing time of 5 minutes removed into distilled water (100-µL drop), and after another standing time of 2 minutes, a 50-µL drop of uranyl-oxalate solution (Ph = 7) was applied to the grids for 5 minutes and covered according to instructions. Afterwards, the grids were washed with distilled water, 2 minutes for each, and examined under a TEM machine. 2.4 Tunable resistive pulse sensing (TRPS) The measurement of nanoparticle size was conducted with the TRPS technique and on the qNano ( Izon Science Ltd). Data analysis was conducted through Izon Control Suite v.3.3.2.2000 (ibid). 2.5 Immunoblotting After being resolved by SDS-PAGE, exosomal proteins of equal amounts were removed to PVDF membranes, which were treated with a solution (5% milk in Tris-buffered saline containing 0.1% Tween 20) for 1 hour at 4°C in dark environments with rabbit primary antibodies against CD81, TSG101, and GM130. Afterwards, they were incubated with HRP-conjugated secondary antibodies for 1 hour at normal room temperature. ECL blotting detection reagents were eventually applied. 2.6 Target genes and pathway TCGA ( Https://portal.gdc.cancer.gov ) 2.7 RNA isolation and real-time PCR TRIzol reagent was selected to extract aggregate RNAs, which were then reversely-transcribed into complementary DNA (cDNA) with the Mix-X miRNA First-Strand Synthesis Kit. Real-time PCR was conducted using TB-Green Premix Ex Taq II Reagent. U6 was viewed as an internal control ( 24 ). All these procedures were performed in accordance with the specific manufacturer’s instructions. For each sample, analysis was made in duplicate. In addition, the evaluation of PCR reaction was by means of melting curves and that of relative quantification of miRNA expression was through the ΔCT method (CtmiRNA-CtU6) as delineated in the previous section ( 25 ). 2.8 Statistical analysis For statistical analysis, SPSS 22.0 and GraphPad Prism 6.0 were selected. Mann–Whitney U or t-test were used for comparison and a paired t-test was performed to compare paired values. As for multiple comparisons, one-way ANOVA was performed. The corresponding cutoff points were determined through ROC curves, where p-value < 0.05 was considered to be of statistical significance. Plus, miRNA or miRNAs in combination were determined after analysis. Results 3.1 Characterization of isolated exosomes Serum exosomes from healthy donors and CRC patients were identified using TEM, qNano and immunoblotting. The microvesicles obtained were cup-shaped, under 150 nm in diameter, consistent with exosomal morphology by TEM (Fig. 1 A). The qNano analysis was employed to quantify the diameter of a single particle, which was detected to be rounded particle with a diameter of 30 to 150 nm (Fig. 1 B). As shown in Fig. 1 C, TSG101 and CD81 were positive under Western blotting, mainly were enriched in exosomes. GM130 is the matrix protein regulating the structure of GA, hence generally employed as a negative control for exosome( 26 ), which found close expression in the cell. The above evidence showed that the small vesicles centrifuged from serum were exosomes considering their size and expression level of marker protein. 3.2 Differential expression of miR-205-5p in pan-cancer To investigate the distribution of miR-205-5p in the human body, we analyzed data from the TCGA RNA seq data. Our analysis revealed that miR-205-5p was primarily located in the lung, brain, stomach, and colorectum, and significant differences in its expression exist between CRC patients with and healthy donors (Figure.2A). Subsequently, we performed a more in-depth analysis of miR-205-5p expression in various types of cancer by analyzing the TCGA RNA-seq data. We observed significant differences in the expression of miR-205-5p across 33 types of cancer using paired or unpaired samples, with the exception of samples lacking normal tissue data. miR-205-5p was considerably downregulated in BRCA (breast invasive carcinoma), COAD (colon adenocarcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma). miR-205-5p expression was increased in BLCA (bladder urothelial carcinoma), CESC (Cervical Cancer), ESCA (esophageal carcinoma), HNSC C (head and neck squamous cell carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), UCEC (uterine corpus endometrial carcinoma) (Fig. 2 B). Finally, the diagnostic efficacy of miR-205-5p was analyzed, in which the the area under the ROC curves (AUCs) of LUSC, CESC, UCEC, READ, COAD, BLCA were 0.97, 0.95, 0.93, 0.89, 0.84, and 0.81, respectively. 3.3 Identification of miR-205-5p in CRC To verify the differential expression of miR-205-5p in CRC, we conducted an analysis of the TCGA database. The results revealed a significant down-regulation of miR-205-5p in CRC compared to healthy donors (Fig. 3 A), as illustrated in the Fig. 3 B, the AUC of miR-205-5p was 0.896. Adjacent tissues versus cancer tissues from GSE49246, discovered that miR-205-5p was remarkably down-regulated in cancer tissues (Fig. 3 C). To investigate whether miR-205-5p was diagnostic of early CRC via TCGA database, we conducted a further analysis of the differential expression between stage I CRC patients and healthy donors (Fig. 3 D), revealing a statistically significant difference. The AUC was calculated to be 0.818(Fig. 3 E). After conducting an analysis on the relationship between miR-205-5p and clinical characteristics using TCGA data, it was observed that miR-205-5p showed no correlation with age, CEA, or metastasis(Figure 4 A, 4 B and 4 F). Nevertheless, it exhibited significant variation when considering different BMI index, pathologic T-stage, and pathologic N-stage(Figure 4 C-E). Specifically, miR-205-5p displayed significant difference in pathologic staging betweenⅠ+Ⅱand Ⅲ+Ⅳ(Fig. 4 G). Furthermore, it was observed that patients with colon polyps had significantly higher expression levels of miR-205-5p compared to those without polyps (Fig. 4 H). Additionally, a lower expression of miR-205-5p was found in patients with progressive disease (PD)and stable disease(SD)compared to those with partial response (PR) + complete response (CR) (Fig. 4 I). Considering the aforementioned findings, it can be inferred that miR-205-5p has the potential to serve as a biomarker for the early diagnosis and prognosis of colorectal cancer. 3.4 Enrichment analysis of miR-205-5p in CRC It was predicted that miR-205-5p was involved in the genetic and pathway regulation, providing a direction for exploring the pathogenesis of CRC through bioinformatics. To investigate the target genes of miR-205-5p, as shown in the Fig. 5 A, we performed correlation analysis of miR-205-5p with target genes from the TCGA CRC database. Heatmap illustrating the top 50 target genes negatively associated with miR-205-5p (Fig. 5 B). The Venn diagram illustrates the overlapping set of predicted target genes of miR-205-5p, target genes that exhibit a negative correlation with miR-205-5p, and differentially expressed genes (DEG) identified between healthy donors and patients with CRC retrieved from the TCGA database (Fig. 5 C). We performed Gene Ontology (GO) analysis and observed that miR-205-5p exhibited significant enrichment in various cellular processes. Specifically, the analysis revealed enrichment in golgi vesicle transport, endosomal transport, vesicle tethering complex, vesicle-mediated transport to the plasma membrane, and cadherin binding involved in cell-cell adhesion, etc (Fig. 5 D). Furthermore, miR-205-5p was found to be predominantly enriched in microRNAs in cancer, colorectal cancer, PI3K-AKT signaling pathway, the thyroid hormone signaling pathway and platinum drug resistance, etc (Fig. 5 E). Importantly, we also conducted an analysis of the Hallmark pathway, which exhibited notable enrichment in E2F targets, G2M checkpoint, mitotic spindle and other related pathways (Fig. 5 F). The above research results pointed to a fact that miR-205-5p is associated with cancerous onset and progression of cancer. 3.5 Characterization of Identified Serum Exosomal miR-205-5p. The expression of miR-205-5p in EDS and exosomes was examined. In effect, miR-205-5p expression in exosomes substantially increased in comparison with EDS (Fig. 6 A). What’s more, miR-205-5p expression in exosomes underwent no substantial changes after RNase A treatment (Fig. 6 B), which proved the stability of exosomal miR-205-5p. Put it simply, the data demonstrated that miR-205-5p chiefly existed in the exosomes, serving the role of protecting miRNAs from being degraded by RNases. Notably, exosomes underwent no considerable change in terms of miR-205-5p expression after being exposed to room temperature for a duration of 0, 6, 12, 18, and 24 hours (Fig. 6 C). 3.6 Exosomal miR-205-5p were associated with CRC To explore the potential value of exosomal miRNA for cancer diagnosis, exosomal miR-205-5p expression levels were quantified in a total 157 CRC patients, 135 healthy donors and 20 benign disease by using RT-qPCR. The results in Fig. 7 A suggested that the measured indicator in the serum decreases considerably in the CRC patients(p < 0.0001, respectively)in contrast with healthy donors and benign disease(p < 0.0001, respectively), but not as significant as miRNAs were detected among healthy subjects and benign patients (p = 0.184, respectively). Additionally, we investigated the correlation between miR-205-5p clinicopathological characterist in CRC patients and healthy donors (Table 1 ). The outcomes suggested that they were not associated with age, gender, histology, progression stage, and among others. Generally, ideal clinical diagnostic biomarkers should have excellent sensitivity and specificity. In order to test whether serum exosomal miR-205-5p was an ideal diagnostic marker for CRC, ROC curve analysis was performed to determine its expression level. We found the area under the ROC curve (AUC) of 0.639 (95% CI: 0.576–0.702, p < 0.0001, Fig. 7 B), and the sensitivity and specificity of detection was 53.5% and 69.2%. Furthermore, the diagnostic efficacy was evaluated. As is indicated in Fig. 7 C, early-stage CRC patients showed a lower level of exosomal miR-205-5p than healthy donors and benign disease (p < 0.001 and p < 0.0001, respectively). Next, ROC curves were devised for the evaluation of exosomal miR-205-5p performance in early diagnosis of CRC. As shown in Fig. 7 D, the AUC of miR-205-5p was 0.66 (95% CI: 0.58–0.739) and the sensitivity and specificity were 53.6% and 71.9% respectively. we continued to observe the expression of the molecules before and after surgical resection in 17 CRC patients. We found that the observed indicator substantially increased after the lesion was removed (p = 0.0053, respectively, Fig. 7 E). The above findings suggested that tumor retention was likely to exert a negative impact on the observed indicator, thereby making it a novel biomarker for surgical efficiency monitoring. Discussion Colorectal cancer (CRC) ranks high among the commonest malignant cancers around the globe and poses a big threat to people’s health and life ( 29 ). Hence, it is of great importance to control the progression of the disease at an early stage. Early diagnosis in CRC patients could realize a five-year survival for 90% of the cases ( 30 ). Yet, delayed diagnosis occurs to nearly 60% of CRC patients, which means a survival rate of merely 8–9%. Hence, a sensitive and specific biomarker is an urgent demand for distinguishing patients with colorectal cancer. The outcome of this study indicated that exosomal miR-205-5p considerably downregulated in CRC, making them potential biomarkers for CRC diagnosis. As has been reported, miR-205-5p is involved in the development of various types of cancers ( 31 – 33 ). For example, overexpression of miR-205-5p was detected in patients with non-small cell lung cancer in comparison with the contrast groups ( 34 ), miR-205-5p was significantly downregulated in estrogen receptor-positive breast cancer by targeting to NFIB ( 35 ). Related studies have shown the regulatory role of miR-205-5p in epithelial-mesenchymal transition by targeting PTEN via PI3K/AKT signaling pathway in cisplatin-resistant nasopharyngeal carcinoma cells ( 36 ), miR-205-5p inhibits PTK7, thereby involved in the proliferation, migration and invasion of CRC( 37 ). Similarly, miR-205-5p was modulated by lncRNA NEAT1 to promote CRC cell proliferation and migration through regulating VEGFA signaling pathway ( 38 ). In addition, there is an article report that anti-correlation existed between the expression level of miR-205-5p and BRCA1 and RAD17 targets in HNSCC( 39 ). Therefore, miR-205-59 might be used as putative effective biomarkers for CRC diagnosis. This study validated that a series of evidence that exosomal miR-205-5p might be employed as diagnostic biomarkers for CRC. miR-205-5p exhibited significant downregulation in colorectal cancer compared to healthy donors via TCGA database. exosomal miR-205-5p witnessed considerable downregulation in CRC patients compared to the control subjects. The AUC of exosmal miR-205-5p was 0.639 with the sensitivity of 53.6% and the specificity of 71.3%. The AUC of exosmal miR-205-5p was 0.66 with the sensitivity of 53.6% and the specificity of 71.9% in early stage CRC patients serum exosomal miR-205-5p. It is significantly increased after surgery, indicating it is closely related with tumor occupying. Finally, miR-205-5p mainly existed in serum exosome and not affected by RNase A. Apart from that, the target gene of exosomal miR-205-5p and pathway were predicted by bioinformatics, we found exosomal miR-205-5p had close correlation with cancer. However, the limitations of this study should be pointed out. First, small cohort size did not render sufficient statistic support to the conclusion. In future study, a larger sample size and clinical follow-up for a longer term are expected. Second, we did not take into account the diagnostic efficacy of the exosomal miR-205-5p in combination with the routine tumor biomarkers of CRC, like CEA and CA199, since relative data concerning healthy donors were not collected. We intend to make further exploration of serum exosomal miR-205-5p in terms of mechanism and prognosis. In a nutshell, this study revealed that the expression levels of exosomal miR-205-5p were remarkably lower in CRC patients, including those at the early stage CRC patients. This finding is valuable for CRC diagnosis and is of practical significance for CRC treatment. Therefore, this study provided evidence that exosomal miR-205-5p is a promising diagnostic biomarker for CRC in clinical practice. Declarations Research funding: This work was supported by Shandong Provincial Natural Science Foundation (ZR2020LZL017), National Natural Science Foundation of China (81773237), Scientific and technological Innovation Program of Clinical Medicine (202019054), Major Science & Technology Innovation Project of Shandong Province（2021LCZX04）. Ethics approval A total of 165 CRC patients, 153 healthy donors, and 20 benign disease patients were admitted in the Shandong Cancer Hospital Affiliated to Shandong University from September 2017 to July 2018（Approval number/ID:SDTHEC20171008）. Acknowledgements We thank the public database resources and all those who helped with this research. We wish to thank the editors and reviewers for their helpful comments on this paper. 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NFIB functions as an oncogene in estrogen receptor-positive breast cancer and is regulated by miR-205-5p. Pathol Res Pract. 2020;216(12):153236. Zhang P, Lu X, Shi Z, Li X, Zhang Y, Zhao S, et al. miR-205-5p regulates epithelial-mesenchymal transition by targeting PTEN via PI3K/AKT signaling pathway in cisplatin-resistant nasopharyngeal carcinoma cells. Gene. 2019;710:103-13. Chen S, Wang Y, Su Y, Zhang L, Zhang M, Li X, et al. miR2055p/PTK7 axis is involved in the proliferation, migration and invasion of colorectal cancer cells. Mol Med Rep. 2018;17(5):6253-60. Liu H, Li A, Sun Z, Zhang J, Xu H. Long non-coding RNA NEAT1 promotes colorectal cancer progression by regulating miR-205-5p/VEGFA axis. Hum Cell. 2020;33(2):386-96. Valenti F, Sacconi A, Ganci F, Grasso G, Strano S, Blandino G, et al. The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas. Cancers (Basel). 2019;11(9). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4013931","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276414579,"identity":"b7cfd7c2-bc36-4886-ae36-d216fd682aef","order_by":0,"name":"Yajing Zhao","email":"","orcid":"","institution":"Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yajing","middleName":"","lastName":"Zhao","suffix":""},{"id":276414580,"identity":"6e52eac6-1af2-4658-8617-e8483ca4e3a7","order_by":1,"name":"Yapeng Zhao","email":"","orcid":"","institution":"Qinghai Red Cross Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yapeng","middleName":"","lastName":"Zhao","suffix":""},{"id":276414581,"identity":"d4d0a790-1eb6-463b-a5d7-9f09d339f3bc","order_by":2,"name":"Lisheng Liu","email":"","orcid":"","institution":"Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lisheng","middleName":"","lastName":"Liu","suffix":""},{"id":276414582,"identity":"77ffc089-7bfb-4603-94a6-294122754a28","order_by":3,"name":"Guanghao Li","email":"","orcid":"","institution":"Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guanghao","middleName":"","lastName":"Li","suffix":""},{"id":276414583,"identity":"c5dadc4f-b91f-4308-8e5e-8e8410b4ebf6","order_by":4,"name":"Yawen Wu","email":"","orcid":"","institution":"Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Wu","suffix":""},{"id":276414584,"identity":"0850e785-7dc9-4b7a-afde-d0e22b736a32","order_by":5,"name":"Yanan Cui","email":"","orcid":"","institution":"Weifang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Cui","suffix":""},{"id":276414585,"identity":"03ca2809-ecd7-4446-b8d2-89bc8f08165e","order_by":6,"name":"Li Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACPgbGBoYPDMz8YB4PMVrYgFoYZzAwSzaQoIWBgZmHNC0SyY2Pbf5YS+jOSGB88LaNQd6coBaeg83GuW3pEmY3EpgN57YxGO5sIKSFvbFNOrfhcB1QC5s0bxtDgsEBQlqYGdt/W/w5DLKF/TdxWoC2MDOwgbWwMROnBegXyV6QX848bJacc07CcAMhLfwS6Q8//ACGmNnx5IMf3pTZyBO0BQkAkwEDgwTx6kfBKBgFo2AU4AYAtxI6O5VTo34AAAAASUVORK5CYII=","orcid":"","institution":"Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2024-03-04 18:33:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4013931/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4013931/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52196514,"identity":"b04bddd2-c441-4deb-9e90-9051c8b8f9bf","added_by":"auto","created_at":"2024-03-07 20:01:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1596438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of isolated exosomes. (A)\u003c/strong\u003e TEM images showing that the exosomes extracted from CRC patients was double-layer round vesicles with a diameter of 50-150nm. \u003cstrong\u003e(B)\u003c/strong\u003e The diameter of the exosomes ranging from 50–150 nm was measured using qNano system. \u003cstrong\u003e(C) \u003c/strong\u003eExosomal proteins CD81, TSG101 and GM130, a negative marker of the exosome was verificated by immunoblotting.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/aba6a3c2f531cc874a481560.png"},{"id":52195405,"identity":"47b65a67-aafc-4fcb-b850-7951da9e9ccd","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1515374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of miR-205-5p in pan-cancer\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Distribution of miR-205-5p in the human body. \u003cstrong\u003e(B)\u003c/strong\u003e Differential expression of miR-205-5p in cancers of 33 types. \u003cstrong\u003e(C) \u003c/strong\u003eArea under the curve of miR-205-5p in different cancer types from TCGA database.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/2982d67bf553969c930e18a3.png"},{"id":52195406,"identity":"e86ee4a5-df11-47bf-9f8a-64a32ce572b2","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":490223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of miR-205-5p in CRC. (A)\u003c/strong\u003e The expression levels of miR-205-5p were notably higher in healthy donors as compared to CRC patients via TCGA database. \u003cstrong\u003e(B) \u003c/strong\u003eThe AUC of miR-205-5p was 0.896 in CRC patients with comparison to healthy donors. \u003cstrong\u003e(C) \u003c/strong\u003eMann-Whitney U test indicated that miR-205-5p was significantly decreased in CRC patients compare with healthy donors from GSE49246. \u003cstrong\u003e(D)\u003c/strong\u003e miR-205-5p expression levels in stageⅠCRC were downregulated compared to healthy donors. (E) The AUC of miR-205-5p inⅠstage CRC patients was 0.818 compared to healthy donors.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/15fe62f2ffe9ddf46db67660.png"},{"id":52195403,"identity":"d6dd9c8b-108a-4cea-a478-8136da8faecd","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":388556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe correlation between miR-205-5p clinicopathological characterist in CRC patients from TCGA database. (A) \u003c/strong\u003eAge. \u003cstrong\u003e(B)\u003c/strong\u003eCEA. \u003cstrong\u003e(C)\u003c/strong\u003eBMI. \u003cstrong\u003e(D)\u003c/strong\u003ePathological T-stage. \u003cstrong\u003e(E)\u003c/strong\u003e Pathological N stage. \u003cstrong\u003e(F) \u003c/strong\u003ePathological M stage.\u003cstrong\u003e (G)\u003c/strong\u003e Pathological stage (Ⅰ+ⅡvsⅢ+Ⅳ).\u003cstrong\u003e(H) \u003c/strong\u003eHistory of colon polyps. \u003cstrong\u003e(I)\u003c/strong\u003e Primary therapy outcome (PD+SD vs PR+CR).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/133e307669ee6c515e7550c3.png"},{"id":52195401,"identity":"3acd57cd-6034-45ec-a9fe-db2862098335","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":924994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of miR-205-5p. (A) \u003c/strong\u003eCorrelation analysis of target genes with miR-205-5p, red dots represent positive correlation with miR-205-5p, Green dots represent negative correlation with miR-205-5p. \u003cstrong\u003e(B）\u003c/strong\u003eHeatmap illustrating the top 50 target genes negatively associated with miR-205-5p. (C) The Venn diagram illustrates the overlapping set of predicted target genes of miR-205-5p, target genes that exhibit a negative correlation with miR-205-5p and DEGs. (D) GO analysis. (E) KEGG pathway. (F) Hallmark pathway\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/b97c38fa779b69e763a91d6e.png"},{"id":52195402,"identity":"cecae9ca-0121-4278-a355-f3debfba4ded","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":533892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of serum exosomal miR-205-5p.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Expression levels of miR-205-5p from EXO and EDS \u003cstrong\u003e(B)\u003c/strong\u003eExpression levels of miR-205-5p in exosomes treated with RNase A or RNA in isolation.\u003cstrong\u003e (C)\u003c/strong\u003e Expression levels of the miR-205-5p in exosomes incubated 0h, 6h, 12h, 18h,24h at room temperature (***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/1109ea71c5be6cb1933195cb.png"},{"id":52195407,"identity":"850d3602-72f6-4496-b4df-11ad32eebb90","added_by":"auto","created_at":"2024-03-07 19:53:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":629547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomal miR-205-5p was downregulated in CRC patients.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Mann-Whitney U test showing that exosomal miR-205-5p decreased considerably in CRC patients in contrast with healthy donors and benign disease（***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant).\u003cstrong\u003e (B)\u003c/strong\u003e The AUC of exosomal miR-205-5p for the detection of CRC was 0.639. \u003cstrong\u003e(C)\u003c/strong\u003e Mann-Whitney U test indicated that exosomal miR-205-5p was significantly decreased in early CRC patients (I=16, II=53) compare with healthy donors (n=135) and benign disease (n=20) （***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant). \u003cstrong\u003e(D)\u003c/strong\u003e The AUC of exosomal miR-205-5p for the detection of early stage CRC was 0.66.\u003cstrong\u003e(F) \u003c/strong\u003eThe serum exosomal miR-205-5p levels was remarkably higher after operation than before operation (**p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/3702c0ed1a22e472e62566c3.png"},{"id":52847213,"identity":"c5835819-f612-45fc-b1e7-d475babb3b0e","added_by":"auto","created_at":"2024-03-17 18:38:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2534363,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4013931/v1/7563294e-7646-43f7-a15b-4ec1184358eb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) ranks third among the commonest cancers and poses a big threat to people\u0026rsquo;s health and life. Nevertheless, approximately four out of ten CRC patients end up with relapse and recurrent metastasis, and merely a small portion of them have a survival time of five years (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Hence, the development of effective and easy-to-use screening tools is of vital importance for early diagnosis and precise stage classification in CRC patients. These may include fluid biopsy such as proteins, tumor markers, circulating tumor cells(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), cell-free DNA(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), and exosomes(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), to facilitate the early detection and precise stage classification of CRC(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExosomes, also referred to as extracellular vesicles, are secreted organelles with a single membrane and measure 30 to 200 nm in diameter (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Cells communicate via exosomes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Therefore, as an essential messenger between cells, exosomes transmit signals to neighbouring cells or to distant anatomical sites by carrying cytokines (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), modify proteic and genetic expression in receiving cells, and thus influence the functions of receiving cells. Exosomes play multi-roles as well in the pathogenesis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) metastasis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and immunity (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Exosomes encompass a variety of proteins (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), miRNAs (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and DNA(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), which are apt to be transferred to target cells. For instance, extensive deregulation of miRNAs have shown in human cancers, which highlights their key role in the onset, development and metastasis of the tumor, and hence miRNAs become potential biomarkers for cancers.\u003c/p\u003e \u003cp\u003eMicroRNA (miRNA) are tiny, non-coding RNA which are generally 20\u0026ndash;25 nucleotide long sequences(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and which will unite with the target mRNA sequence after they enter the receptor cell (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). A host of studies have demonstrated that exosomal miRNA is capable of mediating communication between cells, thus involved in tumor carcinogenesis, metastasis, immunosuppression, angiogenesis, and other aspects (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Emerging evidence suggested that five types of exosomal miRNAs, namely miR-205, miR-19a, miR-19b, miR-30b and miR-20a, are promising diagnostic markers of squamous cell lung cancer due to their decreased levels of circulation after resection operation of the lesion (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The level of serum exosomal miR-205 was up-regulated in ovarian cancer tissues, and its over-expression was linked to cancerous metastasis in ovarian carcinoma patients (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The relative expressions of exosomal miR-1246 in serum were signifcantly overexpressed in gastric cancer patients compared to that in healthy controls (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Nasopharyngeal carcinoma cells discharge exosomes that contain certain types of miRNAs that play an inhibitory role in T-cell proliferation,targeting MAPK-1 and STAT pathways (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe increase of neutrophil count in peripheral blood has been shown to be related to poor clinical outcomes in pancreatic cancer, gastric cancer and breast cancer. Rao et al demonstrated that the increase in intertumoral neutrophils was associated with malignant phenotypes and could predict adverse prognosis in CRC.\u003c/p\u003e \u003cp\u003eThis study aimed to verify the different expression levels of serum exosomal miRNA in CRC patients and in contrast groups by means of small RNA sequencing and fluorescence quantitative PCR. Consequently, exosomal miR-205-5p were chosen and their related with diagnostic efficiency and clinical features was analyzed. Therefore, exosomal miR-205-5p as a novel diagnostic biomarker.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003e2.1 Patients and clinical samples\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the study, we selected altogether 157 CRC patients, 135 healthy donors and 20 benign disease patients hospitalized at the Shangdong Caner Hospital in September 2017 to July 2018. We obtained their written consent. The estimation of tumor staging was performed in accordance with the AJCC Cancer Staging Handbook, 2017. The protocol was approved by the Shandong Cancer Hospital Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences of committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki. No patient was given anti-tumor treatment prior to serum collection and no patient was diagnosed with diseases of the endocrine, immune or metabolic system. Sera collection was performed on 17/157 patients with CRC, who received surgical treatment after two months. Patients\u0026rsquo; clinical features and diabetic history were investigated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of CRC patients for differentially expressed exosomal miR-205-5p\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003emiR-205-5p\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. cases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian with interquartile range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.4729(4.0255\u0026ndash;4.9203)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.954\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.4001(3.9076\u0026ndash;4.8925)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.6445(4.2411\u0026ndash;5.0479)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0158(3.4443\u0026ndash;4.5872)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrinking status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0204(3.0903\u0026ndash;4.9504)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.5265(4.1756\u0026ndash;4.8774\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiabetes status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.3734(3.2583\u0026ndash;5.4885)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.951\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.4449(4.0973\u0026ndash;4.7924)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor position\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.4328(3.9982\u0026ndash;4.8674)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.920\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecolon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.4411(3.9231\u0026ndash;4.9591)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.5427(3.9112\u0026ndash;5.1742)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.748\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.3591(3.8134\u0026ndash;4.9048)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eunknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.3886(3.9132\u0026ndash;4.8640)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.405\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.6332 (4.1118\u0026ndash;5.1546)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eunknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.1779(3.4966\u0026ndash;4.8591)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.5104(4.1304\u0026ndash;4.8903)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Isolation of exosomes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSerum underwent ultracentrifugation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) under the condition of 10000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C for the purpose of removing the cellular debris, and then under the condition of 100,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 2 h at 4\u0026deg;C for exosome precipitation. Then, the samples were analyzed using the following techniques.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 TEM assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM) was conducted for the purpose of identifying the purified exosomes. The exosome pellets were moved to the grids of solution (a 50 \u0026micro;L drop of 1% glutaraldehyde), and after a standing time of 5 minutes removed into distilled water (100-\u0026micro;L drop), and after another standing time of 2 minutes, a 50-\u0026micro;L drop of uranyl-oxalate solution (Ph\u0026thinsp;=\u0026thinsp;7) was applied to the grids for 5 minutes and covered according to instructions. Afterwards, the grids were washed with distilled water, 2 minutes for each, and examined under a TEM machine.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 Tunable resistive pulse sensing (TRPS)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe measurement of nanoparticle size was conducted with the TRPS technique and on the qNano ( Izon Science Ltd). Data analysis was conducted through Izon Control Suite v.3.3.2.2000 (ibid).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 Immunoblotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter being resolved by SDS-PAGE, exosomal proteins of equal amounts were removed to PVDF membranes, which were treated with a solution (5% milk in Tris-buffered saline containing 0.1% Tween 20) for 1 hour at 4\u0026deg;C in dark environments with rabbit primary antibodies against CD81, TSG101, and GM130. Afterwards, they were incubated with HRP-conjugated secondary antibodies for 1 hour at normal room temperature. ECL blotting detection reagents were eventually applied.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6 Target genes and pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTCGA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eHttps://portal.gdc.cancer.gov\u003c/span\u003e\u003cspan address=\"http://Https://portal.gdc.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.7 RNA isolation and real-time PCR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTRIzol reagent was selected to extract aggregate RNAs, which were then reversely-transcribed into complementary DNA (cDNA) with the Mix-X miRNA First-Strand Synthesis Kit. Real-time PCR was conducted using TB-Green Premix Ex Taq II Reagent. U6 was viewed as an internal control (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). All these procedures were performed in accordance with the specific manufacturer\u0026rsquo;s instructions. For each sample, analysis was made in duplicate. In addition, the evaluation of PCR reaction was by means of melting curves and that of relative quantification of miRNA expression was through the ΔCT method (CtmiRNA-CtU6) as delineated in the previous section (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.8 Statistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor statistical analysis, SPSS 22.0 and GraphPad Prism 6.0 were selected. Mann\u0026ndash;Whitney U or t-test were used for comparison and a paired t-test was performed to compare paired values. As for multiple comparisons, one-way ANOVA was performed. The corresponding cutoff points were determined through ROC curves, where p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be of statistical significance. Plus, miRNA or miRNAs in combination were determined after analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e3.1 Characterization of isolated exosomes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSerum exosomes from healthy donors and CRC patients were identified using TEM, qNano and immunoblotting. The microvesicles obtained were cup-shaped, under 150 nm in diameter, consistent with exosomal morphology by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The qNano analysis was employed to quantify the diameter of a single particle, which was detected to be rounded particle with a diameter of 30 to 150 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, TSG101 and CD81 were positive under Western blotting, mainly were enriched in exosomes. GM130 is the matrix protein regulating the structure of GA, hence generally employed as a negative control for exosome(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), which found close expression in the cell. The above evidence showed that the small vesicles centrifuged from serum were exosomes considering their size and expression level of marker protein.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Differential expression of miR-205-5p in pan-cancer\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the distribution of miR-205-5p in the human body, we analyzed data from the TCGA RNA seq data. Our analysis revealed that miR-205-5p was primarily located in the lung, brain, stomach, and colorectum, and significant differences in its expression exist between CRC patients with and healthy donors (Figure.2A). Subsequently, we performed a more in-depth analysis of miR-205-5p expression in various types of cancer by analyzing the TCGA RNA-seq data. We observed significant differences in the expression of miR-205-5p across 33 types of cancer using paired or unpaired samples, with the exception of samples lacking normal tissue data. miR-205-5p was considerably downregulated in BRCA (breast invasive carcinoma), COAD (colon adenocarcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma). miR-205-5p expression was increased in BLCA (bladder urothelial carcinoma), CESC (Cervical Cancer), ESCA (esophageal carcinoma), HNSC C (head and neck squamous cell carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), UCEC (uterine corpus endometrial carcinoma) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Finally, the diagnostic efficacy of miR-205-5p was analyzed, in which the the area under the ROC curves (AUCs) of LUSC, CESC, UCEC, READ, COAD, BLCA were 0.97, 0.95, 0.93, 0.89, 0.84, and 0.81, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Identification of miR-205-5p in CRC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo verify the differential expression of miR-205-5p in CRC, we conducted an analysis of the TCGA database. The results revealed a significant down-regulation of miR-205-5p in CRC compared to healthy donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), as illustrated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the AUC of miR-205-5p was 0.896. Adjacent tissues versus cancer tissues from GSE49246, discovered that miR-205-5p was remarkably down-regulated in cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo investigate whether miR-205-5p was diagnostic of early CRC via TCGA database, we conducted a further analysis of the differential expression between stage I CRC patients and healthy donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), revealing a statistically significant difference. The AUC was calculated to be 0.818(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eAfter conducting an analysis on the relationship between miR-205-5p and clinical characteristics using TCGA data, it was observed that miR-205-5p showed no correlation with age, CEA, or metastasis(Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Nevertheless, it exhibited significant variation when considering different BMI index, pathologic T-stage, and pathologic N-stage(Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). Specifically, miR-205-5p displayed significant difference in pathologic staging betweenⅠ+Ⅱand Ⅲ+Ⅳ(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Furthermore, it was observed that patients with colon polyps had significantly higher expression levels of miR-205-5p compared to those without polyps (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Additionally, a lower expression of miR-205-5p was found in patients with progressive disease (PD)and stable disease(SD)compared to those with partial response (PR)\u0026thinsp;+\u0026thinsp;complete response (CR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Considering the aforementioned findings, it can be inferred that miR-205-5p has the potential to serve as a biomarker for the early diagnosis and prognosis of colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Enrichment analysis of miR-205-5p in CRC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt was predicted that miR-205-5p was involved in the genetic and pathway regulation, providing a direction for exploring the pathogenesis of CRC through bioinformatics. To investigate the target genes of miR-205-5p, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, we performed correlation analysis of miR-205-5p with target genes from the TCGA CRC database. Heatmap illustrating the top 50 target genes negatively associated with miR-205-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The Venn diagram illustrates the overlapping set of predicted target genes of miR-205-5p, target genes that exhibit a negative correlation with miR-205-5p, and differentially expressed genes (DEG) identified between healthy donors and patients with CRC retrieved from the TCGA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We performed Gene Ontology (GO) analysis and observed that miR-205-5p exhibited significant enrichment in various cellular processes. Specifically, the analysis revealed enrichment in golgi vesicle transport, endosomal transport, vesicle tethering complex, vesicle-mediated transport to the plasma membrane, and cadherin binding involved in cell-cell adhesion, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, miR-205-5p was found to be predominantly enriched in microRNAs in cancer, colorectal cancer, PI3K-AKT signaling pathway, the thyroid hormone signaling pathway and platinum drug resistance, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Importantly, we also conducted an analysis of the Hallmark pathway, which exhibited notable enrichment in E2F targets, G2M checkpoint, mitotic spindle and other related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The above research results pointed to a fact that miR-205-5p is associated with cancerous onset and progression of cancer.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Characterization of Identified Serum Exosomal miR-205-5p.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression of miR-205-5p in EDS and exosomes was examined. In effect, miR-205-5p expression in exosomes substantially increased in comparison with EDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). What\u0026rsquo;s more, miR-205-5p expression in exosomes underwent no substantial changes after RNase A treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), which proved the stability of exosomal miR-205-5p. Put it simply, the data demonstrated that miR-205-5p chiefly existed in the exosomes, serving the role of protecting miRNAs from being degraded by RNases. Notably, exosomes underwent no considerable change in terms of miR-205-5p expression after being exposed to room temperature for a duration of 0, 6, 12, 18, and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 Exosomal miR-205-5p were associated with CRC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential value of exosomal miRNA for cancer diagnosis, exosomal miR-205-5p expression levels were quantified in a total 157 CRC patients, 135 healthy donors and 20 benign disease by using RT-qPCR. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA suggested that the measured indicator in the serum decreases considerably in the CRC patients(p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively)in contrast with healthy donors and benign disease(p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), but not as significant as miRNAs were detected among healthy subjects and benign patients (p\u0026thinsp;=\u0026thinsp;0.184, respectively).\u003c/p\u003e \u003cp\u003eAdditionally, we investigated the correlation between miR-205-5p clinicopathological characterist in CRC patients and healthy donors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The outcomes suggested that they were not associated with age, gender, histology, progression stage, and among others.\u003c/p\u003e \u003cp\u003eGenerally, ideal clinical diagnostic biomarkers should have excellent sensitivity and specificity. In order to test whether serum exosomal miR-205-5p was an ideal diagnostic marker for CRC, ROC curve analysis was performed to determine its expression level. We found the area under the ROC curve (AUC) of 0.639 (95% CI: 0.576\u0026ndash;0.702, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), and the sensitivity and specificity of detection was 53.5% and 69.2%.\u003c/p\u003e \u003cp\u003eFurthermore, the diagnostic efficacy was evaluated. As is indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, early-stage CRC patients showed a lower level of exosomal miR-205-5p than healthy donors and benign disease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). Next, ROC curves were devised for the evaluation of exosomal miR-205-5p performance in early diagnosis of CRC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, the AUC of miR-205-5p was 0.66 (95% CI: 0.58\u0026ndash;0.739) and the sensitivity and specificity were 53.6% and 71.9% respectively.\u003c/p\u003e \u003cp\u003ewe continued to observe the expression of the molecules before and after surgical resection in 17 CRC patients. We found that the observed indicator substantially increased after the lesion was removed (p\u0026thinsp;=\u0026thinsp;0.0053, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The above findings suggested that tumor retention was likely to exert a negative impact on the observed indicator, thereby making it a novel biomarker for surgical efficiency monitoring.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eColorectal cancer (CRC) ranks high among the commonest malignant cancers around the globe and poses a big threat to people\u0026rsquo;s health and life (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Hence, it is of great importance to control the progression of the disease at an early stage. Early diagnosis in CRC patients could realize a five-year survival for 90% of the cases (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Yet, delayed diagnosis occurs to nearly 60% of CRC patients, which means a survival rate of merely 8\u0026ndash;9%. Hence, a sensitive and specific biomarker is an urgent demand for distinguishing patients with colorectal cancer. The outcome of this study indicated that exosomal miR-205-5p considerably downregulated in CRC, making them potential biomarkers for CRC diagnosis.\u003c/p\u003e \u003cp\u003eAs has been reported, miR-205-5p is involved in the development of various types of cancers (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). For example, overexpression of miR-205-5p was detected in patients with non-small cell lung cancer in comparison with the contrast groups (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), miR-205-5p was significantly downregulated in estrogen receptor-positive breast cancer by targeting to NFIB (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Related studies have shown the regulatory role of miR-205-5p in epithelial-mesenchymal transition by targeting PTEN via PI3K/AKT signaling pathway in cisplatin-resistant nasopharyngeal carcinoma cells (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), miR-205-5p inhibits PTK7, thereby involved in the proliferation, migration and invasion of CRC(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Similarly, miR-205-5p was modulated by lncRNA NEAT1 to promote CRC cell proliferation and migration through regulating VEGFA signaling pathway (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In addition, there is an article report that anti-correlation existed between the expression level of miR-205-5p and BRCA1 and RAD17 targets in HNSCC(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Therefore, miR-205-59 might be used as putative effective biomarkers for CRC diagnosis.\u003c/p\u003e \u003cp\u003eThis study validated that a series of evidence that exosomal miR-205-5p might be employed as diagnostic biomarkers for CRC. miR-205-5p exhibited significant downregulation in colorectal cancer compared to healthy donors via TCGA database. exosomal miR-205-5p witnessed considerable downregulation in CRC patients compared to the control subjects. The AUC of exosmal miR-205-5p was 0.639 with the sensitivity of 53.6% and the specificity of 71.3%. The AUC of exosmal miR-205-5p was 0.66 with the sensitivity of 53.6% and the specificity of 71.9% in early stage CRC patients serum exosomal miR-205-5p. It is significantly increased after surgery, indicating it is closely related with tumor occupying. Finally, miR-205-5p mainly existed in serum exosome and not affected by RNase A. Apart from that, the target gene of exosomal miR-205-5p and pathway were predicted by bioinformatics, we found exosomal miR-205-5p had close correlation with cancer.\u003c/p\u003e \u003cp\u003eHowever, the limitations of this study should be pointed out. First, small cohort size did not render sufficient statistic support to the conclusion. In future study, a larger sample size and clinical follow-up for a longer term are expected. Second, we did not take into account the diagnostic efficacy of the exosomal miR-205-5p in combination with the routine tumor biomarkers of CRC, like CEA and CA199, since relative data concerning healthy donors were not collected. We intend to make further exploration of serum exosomal miR-205-5p in terms of mechanism and prognosis.\u003c/p\u003e \u003cp\u003eIn a nutshell, this study revealed that the expression levels of exosomal miR-205-5p were remarkably lower in CRC patients, including those at the early stage CRC patients. This finding is valuable for CRC diagnosis and is of practical significance for CRC treatment. Therefore, this study provided evidence that exosomal miR-205-5p is a promising diagnostic biomarker for CRC in clinical practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eResearch funding:\u003c/strong\u003e This work was supported by Shandong Provincial Natural Science Foundation (ZR2020LZL017), National Natural Science Foundation of China (81773237), Scientific and technological Innovation Program of Clinical Medicine (202019054), Major Science \u0026amp; Technology Innovation Project of Shandong Province（2021LCZX04）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 165 CRC patients, 153 healthy donors, and 20 benign disease patients were admitted in the Shandong Cancer Hospital Affiliated to Shandong University from September 2017 to July 2018（Approval number/ID:SDTHEC20171008）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the public database resources and all those who helped with this research. We wish to thank the editors and reviewers for their helpful comments on this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eNo Data associated in the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiyamoto Y, Hayashi N, Sakamoto Y, Ohuchi M, Tokunagam R, Kurashige J, et al. Predictors of long-term survival in patients with stage IV colorectal cancer with multi-organ metastases: a single-center retrospective analysis. Int J Clin Oncol. 2015;20(6):1140-6.\u003c/li\u003e\n\u003cli\u003eTsikitis VL, Larson DW, Huebner M, Lohse CM, Thompson PA. Predictors of recurrence free survival for patients with stage II and III colon cancer. 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Gene. 2019;710:103-13.\u003c/li\u003e\n\u003cli\u003eChen S, Wang Y, Su Y, Zhang L, Zhang M, Li X, et al. miR2055p/PTK7 axis is involved in the proliferation, migration and invasion of colorectal cancer cells. Mol Med Rep. 2018;17(5):6253-60.\u003c/li\u003e\n\u003cli\u003eLiu H, Li A, Sun Z, Zhang J, Xu H. Long non-coding RNA NEAT1 promotes colorectal cancer progression by regulating miR-205-5p/VEGFA axis. Hum Cell. 2020;33(2):386-96.\u003c/li\u003e\n\u003cli\u003eValenti F, Sacconi A, Ganci F, Grasso G, Strano S, Blandino G, et al. The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas. Cancers (Basel). 2019;11(9).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exosomes, colorectal cancer, miR-205-5p, diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-4013931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4013931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Tumor-exosomal miRNA play crucial roles in cancer diagnosis. The current reports aimed to found an exosomal miRNA for colorectal cancer(CRC) with non-invasiveness, sensitivity and specificity.\u003c/p\u003e\n\u003cp\u003ePatients and methods: The exosome was extracted from CRC patients and healthy donors using ultracentrifugation to verify by TEM, qNano and western blot. Differential expression level and clinical characterization of miR-205-5p were analyzed in colorectal cancer via TCGA. Real Time Quantitative PCR was employed to determine the different expression levels of exosomal miRNAs in 157 primary CRC patients and 135 healthy donors. Predictions were made concerning target genes to determine the direction for further exploring the etiopathogenesis of the disease by means of bioinformatics.\u003c/p\u003e\n\u003cp\u003eResults: The expression of miR-205-5p demonstrated a substantial downregulation in colorectal cancer compared to healthy donors, as determined through analysis of the TCGA database. We conducted a prediction and analysis of the functional enrichment of downstream target genes regulated by miR-205-5p. Reduced level of exosomal miR-205-5p in serum from CRC patients was found compared with healthy controls (p\u0026lt;0.0001, respectively) and benign disease (p\u0026lt;0.0001, respectively). The levels of expression of exosomal miR-205-5p were substantially lower in early stage CRC patients than in the contrast groups (p\u0026lt;0.0001, respectively). The expression levels of exosomal miR-205-5p saw significant increase postoperatively (p=0.0053, respectively).\u003c/p\u003e\n\u003cp\u003eConclusions: The present study demonstrated that serum exosomal miR-205-5p may be a diagnostic biomarker for colorectal cancer.\u003c/p\u003e","manuscriptTitle":"Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 19:52:58","doi":"10.21203/rs.3.rs-4013931/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"093f8d04-5529-4e14-a6da-e86c7ae9e94e","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T18:29:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 19:52:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4013931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4013931","identity":"rs-4013931","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}